The large-scale, economic purification of proteins is increasingly an important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors. The protein of interest must be isolated from the mixture of compounds fed to the cells and from the by-products of the cells themselves (feed stream) to purity sufficient for use as a human therapeutic. The standards set by health authorities for proteins intended for human administration regarding impurities from the feed stream are very high. Many purification methods for proteins known in the art contain steps requiring the application e.g. of low or high pH, high salt concentration or other extreme conditions that may irreversibly jeopardize the biological activity of the protein to be purified and are therefore not suitable. Thus, separation of the desired protein to sufficient purity poses a formidable challenge. Historically, protein purification schemes have been predicated on differences in the molecular properties of size, charge and solubility between the protein to be purified and undesired protein contaminants. Protocols based on these parameters include size exclusion chromatography, ion exchange chromatography, differential precipitation and the like.
Antibodies and antibody fragments are of increasing importance in a range of therapeutic areas. One of the most important methods of producing antibody fragments is by recombinant technology. Such techniques use a host cell to express the desired antibody, or antibody fragment, which is then separated from the production medium and purified.
Antibodies require glycosylation and are therefore generally expressed in eukaryotic expression systems employing eukaryotic cells, in particular mammalian cells such as CHO, PER. C6, NSO, BHK or Sp2/0 cells. In eukaryotic expression systems the protein of interest expressed such as an antibody is generally secreted into the cell culture medium. The medium can subsequently be separated easily from the protein secreting cells, e.g. by centrifugation or filtration.
Almost all current industrial antibody purification platforms use protein A (described e.g. in WO 98/23645). Protein A is a cell surface protein found in the cell wall of the bacteria staphylococcus aureus that binds to the Fc portion of mammalian immunoglobulin. Protein A has a high affinity to human IgG1 and IgG2 and a moderate affinity to human IgM, IgA and IgE antibodies. Consequently, protein A purification is not well suited for antibody fragments that lack the Fc portion.
A protein that does not require glycosylation is preferably expressed in prokaryotic expression systems employing prokaryotic cells such as gram-negative bacteria. Particularly, an antibody that does not require glycosylation, for example an antibody fragment such as a Fab, a Fab′ or an scFv is preferably expressed in such systems. Prokaryotic expression systems and in particular Escherichia coli (E. coli) systems or other gram-negative bacteria allow the manufacturing of proteins that do not require glycosylation, such as antibody fragments, in an economically attractive way. Manufacturing of proteins in E. coli is beneficial in particular due to due to lower costs of goods and faster drug development processes (Humphreys, 2003; Humphreys, 2003). Prokaryotic and in particular E. coli protein expression systems are well known in the art (Swartz, 2001; Jana and Deb, 2005; Terpe, 2006). Prokaryotic cells do not actively secrete a heterologous protein of interest expressed in the cell. Gram-negative prokaryotic cells such as E. coli, however, can be engineered such that heterologous proteins expressed in the cell, such as antibody fragments, are exported into the periplasmic space where they can form disulfide bonds. Isolation of these heterologous proteins from the periplasmic space requires the disruption of the outer membrane of the prokaryotic cells which results in substantial release also of host cell proteins (HCPs). Methods for disrupting the outer membrane of a gram-negative prokaryotic cell and subsequent harvest of the cell culture fluid containing the heterologous are well known in the art. Manufacturing of antibody fragments in E. coli also results in the production of by-products such as truncated light chains, glutathione adducts of light chains and light chain dimers (Battersby et al., 2001).
Cell culture fluid (feed stream) harvested from prokaryotic expression systems such as E. coli expression systems and in particular periplasmic cell extract from gram-negative bacteria differs substantially from cell culture fluid harvested from eukaryotic expression systems in the relative amount and composition of HCPs, bacterial DNA and endotoxin which need to be separated from the heterologous protein of interest that is expressed in the prokaryotic or eukaryotic expression system. Concentration of HCP as well as complexity and heterogeneity of HCP depend on the expression system or cell line and the cell culture conditions (Arunakumari, 2007). Purification of antibody fragments expressed in prokaryotic expression systems, in particular in gram-negative prokaryotic expression systems, faces therefore a different set of challenges and requires different approaches (Humphreys and Glover, 2001). Basic principles of purification of monoclonal antibody fragments are known in the art (Spitali, 2009). There are two medicinal products currently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) which comprise an antibody fragment as active ingredient which is produced in microbial cells: certolizumab pegol (Cimzia®) comprises a Fab binding specifically to TNFα and ranibizumab (Lucentis®) is a Fab fragment binding specifically to vascular endothelial growth factor (VEGF). Purification of ranibizumab from microbial feed stream is performed using a process with four chromatography steps (Walsh, 2007). The medicinal product abciximab (ReoPro®) comprises the Fab fragment of the chimeric human-murine monoclonal antibody 7E3 which binds to the glycoprotein (GP) IIb/IIIa receptor of human platelets and inhibits platelet aggregation. The chimeric 7E3 antibody is produced by continuous perfusion in mammalian cell culture. The 48 Kd Fab fragment is obtained from the purified full length antibody after digestion with papain and column chromatography.
Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) of interest and a specific ligand coupled to a chromatography matrix. The interaction between the protein of interest and ligand coupled to the chromatography matrix can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Affinity purification requires a biospecific ligand that can be covalently attached to a chromatography matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Despite its common use, affinity chromatography is costly, particularly at the industrial scale necessary to purify therapeutic proteins.
Ion exchange chromatography can be used to purify ionizable molecules. Ionized molecules are separated on the basis of the non-specific electrostatic interaction of their charged groups with oppositely charged molecules attached to the solid phase support matrix, thereby retarding those ionized molecules that interact more strongly with solid phase. The net charge of each type of ionized molecule, and its affinity for the matrix, varies according to the number of charged groups, the charge of each group, and the nature of the molecules competing for interaction with the charged solid phase matrix. These differences result in resolution of various molecule types by ion-exchange chromatography. Elution of molecules that are bound to the solid phase is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). Two general types of interaction are known: Anionic exchange chromatography mediated by negatively charged amino acid side chains (e.g. aspartic acid and glutamic acid) interacting with positively charged surfaces and cationic exchange chromatography mediated by positively charged amino acid residues (e.g. lysine and arginine) interacting with negatively charged surfaces. Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, loses its charge at high pH. Diethylaminoethyl (DEAE)-cellulose is an example of a weak anion exchanger, where the amino group can be positively charged below pH˜9 and gradually loses its charge at higher pH values. DEAE or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, for instance.
An alternative to elution by increase in ion strength of the elution buffer (elution chromatography) is elution using molecules which have a higher dynamic affinity for the stationary phase than the bound protein. This mode of performing ion-exchange chromatography is called displacement chromatography. Displacement chromatography is fundamentally different from any other modes of chromatography in that the solutes are not desorbed in the mobile phase modifier and separated by differences in migration rates (Tugcu, 2008). In displacement, molecules are forced to migrate down the chromatographic column by an advancing shock wave of a displacer molecule that has a higher affinity for the stationary phase than any component from the feed stream. It is this forced migration that results in higher product concentrations and purities compared to other modes of operation of high retention, followed by a constant infusion of a displacer solution into the column.
Dynamic binding capacity describes the amount of protein of interest which will bind to a chromatography resin in a column under defined flow conditions. The dynamic binding capacity for a chromatography resin is dependent on running conditions (e.g. flow rate, pH and conductivity), origin of the sample, sample preparation and the other binding impurities present. Dynamic binding capacities are determined by loading a sample containing a known concentration of the protein of interest, and monitoring the concentration in the column flow-through (Do et al., 2008). The dynamic binding capacity of an ion exchange resin is defined as the point during loading when the protein of interest starts to be recovered in the flow-through. Typically a value of 10% for the proportion of protein of interest in the flow-through compared to the load is used to define this point (McCue et al., 2003). For impurity removal, the threshold for the impurity in the flow-through is set according to criteria specific to the application.
WO 99/57134, WO 2004/024866 and WO 2007/117490 relate to processes for protein or antibody purification comprising ion exchange chromatography. The processes are exemplified using antibodies produced in mammalian cells. WO 2009/058812 relates to a process for antibody purification comprising cation exchange chromatography. The process is exemplified using antibodies produced in mammalian cells. WO 2007/108955 relates to a two-step non-affinity ion exchange chromatograph process for protein purification comprising cation exchange chromatography followed by ion exchange chromatography. The Example in WO 2007/108955 describes the purification of fully human antibody produced in mammalian cells. Multiple washing steps were performed during cation exchange chromatography and the eluate diluted prior to anion exchange chromatography. Humphreys et al. describes the purification of Fab′ at a laboratory scale using cation exchange chromatography and ion exchange chromatography (Humphreys et al., 2004). WO 2004/035792 relates to the generation of E. coli strains expressing mutant PhoS protein in order to reduce PhoS protein impurities in antibody fragment preparations purified from bacterial cell culture.
CDP870 is a genetically engineered antibody fragment (Fab′) chemically linked to a PEG moiety as described in WO 01/94585 (which is incorporated herein by reference in its entirety). CDP870 has potent human TNFα neutralizing properties.
There is a need in the art for methods of purifying antibody fragments from cell culture fluids harvested from prokaryotic and in particular gram-negative bacteria such as E. coli expression systems. There is particular need in the art for methods of purifying antibody fragments from periplasmic cell extracts harvested from prokaryotic and in particular gram-negative bacteria such as E. coli expression systems that are suitable to operate with cell extracts that contain a very high titer of antibody fragment or HCP or both antibody fragment and HCP. High titer expression of the antibody fragments require methods suitable for the purification of the large quantities of antibody fragments in an economical manner: reducing the column sizes, buffer usage and processing times (GE Healthcare data file 11-0025-76 AE, 2007).